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1.
Neurosci Lett ; 280(3): 223-7, 2000 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-10675801

RESUMEN

5-HT(1) receptor subtypes ((1B), (1D) and (1F)) have been implicated in migraine pathophysiology and their ligands have been examined for pharmacological actions in various experimental animal models. Considerable divergences exist, however, in their primary sequences between experimental animals and human, and additional models closer to human, such as non-human primates seem to be useful for migraine research. Earlier, we cloned the 5-HT(1D), and here 5-HT(1B) and 5-HT(1F) receptors from the chimpanzee, gorilla and Rhesus monkey, via polymerase chain reactions with their genomic DNAs and primers designed from the corresponding human receptors. Direct sequencing of PCR products showed that the 5-HT(1B) receptors from the chimpanzee, gorilla and monkey differ from the human receptor by 0, 1 and 7 residues, respectively while 5-HT(1F) receptors differ by 0, 3 and 10 residues, respectively. These divergent residues are mostly conservatively substituted and also largely confined to the N-terminal region and the 3rd intracellular loop, away from transmembrane segments and intracellular loops near membrane which are critical for ligand binding and G protein coupling. The chimpanzee 5-HT(1D), 5-HT(1B) and monkey 5-HT(1F) receptors, as heterologously expressed in human embryonic kidney 293 cells, showed robust agonist-induced guanosine 5'gamma (35)S triphosphate (GTPgamma(35)S) binding through activation of G proteins containing Ggamma(i) subunits. Moreover, pronounced inhibition of basal GTPgamma(35)S binding by methiothepin (an antagonist), representing constitutively active receptors, was observed with only 5-HT(1D). Overall, ligand binding and GTPgamma(35)S binding profiles for these primate receptors are comparable to those for the human receptors and validate non-human primates as useful models for human migraine research.


Asunto(s)
Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Animales , Clonación Molecular , Gorilla gorilla , Humanos , Cinética , Ligandos , Macaca mulatta , Pan troglodytes , Receptor de Serotonina 5-HT1B , Receptor de Serotonina 5-HT1D , Receptores de Serotonina/genética , Proteínas Recombinantes/metabolismo , Radioisótopos de Azufre , Receptor de Serotonina 5-HT1F
2.
Br J Pharmacol ; 128(6): 1181-8, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10578130

RESUMEN

1 The D3 dopamine receptor presumably activates Gi/Go subtypes of G-proteins, like the structurally analogous D2 receptor, but its signalling targets have not been clearly established due to weak functional signals from cloned receptors as heterologously expressed in mostly non-neuronal cell lines. 2 In this study, recombinant human D3 receptors expressed in a human neuroblastoma cell line, SH-SY5Y, produced much greater signals than those expressed in a human embryonic kidney cell line, HEK293. Quinpirole, a prototypic agonist, markedly inhibited forskolin-stimulated cyclic AMP production and Ca2+-channel (N-type) currents in SH-SY5Y cells, and enhanced GTPgamma35S binding in isolated membranes, nearly ten times greater than that observed in HEK293 cell membranes. 3 GTPgamma35S-bound Galpha subunits from quinpirole-activated and solubilized membranes were monitored upon immobilization with various Galpha-specific antibodies. Galphao subunits (not Galphai) were highly labelled with GTPgamma35S in SH-SY5Y, but not in HEK293 cell membranes, despite their abundance in the both cell types, as shown with reverse transcription-polymerase chain reaction and Western blots. N-type Ca2+ channels and adenylyl cyclase V (D3-specific effector), on the other hand, exist only in SH-SY5Y cells. 4 More efficient coupling of the D3 receptor to Go subtypes in SH-SY5Y than HEK293 cells may be attributed, at least in part, to the two D3 neuronal effectors only present in SH-SY5Y cells (N-type Ca2+-channels and adenylyl cyclase V). The abundance of Go subtypes in the both cell lines seems to indicate their availability not a limiting factor.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Dopamina D2/metabolismo , Adenilil Ciclasas/genética , Unión Competitiva/efectos de los fármacos , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Quinpirol/metabolismo , Quinpirol/farmacología , ARN/genética , ARN/metabolismo , Ensayo de Unión Radioligante , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/fisiología , Receptores de Dopamina D3 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Radioisótopos de Azufre , Tritio , Células Tumorales Cultivadas
3.
J Biol Chem ; 274(42): 29587-90, 1999 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-10514423

RESUMEN

Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8- and 50-kDa chains which make up the active enzyme reside, respectively, at the NH(2)- and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.


Asunto(s)
Precursores Enzimáticos/metabolismo , Glucuronidasa , Glicósido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Plaquetas/enzimología , Cromatografía Líquida de Alta Presión , ADN Complementario , Dimerización , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Humanos , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
4.
Br J Pharmacol ; 127(2): 468-72, 1999 May.
Artículo en Inglés | MEDLINE | ID: mdl-10385247

RESUMEN

1. Both the 5-HT1D and 5-HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5-HT1D receptors with much higher affinity than 5-HT1B receptors. In the guinea-pig, a primary animal model for anti-migraine drug testing, however, isochromans bound the 5-HT1D receptor with lower affinity than the gorilla receptor. 2. This species-specific pharmacology was investigated, using site-directed mutagenesis on cloned guinea-pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea-pig 5-HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. 3. For G protein-coupling, serotonin marginally enhanced GTPgamma35S binding in membranes expressing the guinea-pig 5-HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPgamma35S binding in the latter nearly as much as serotonin, and several isochromans by 30-60% of serotonin. 4. We discovered key differences in the function and binding properties of guinea-pig and gorilla 5-HT1D receptors, and identified contributions of I100 and H102 of primate 5-HT1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.


Asunto(s)
Cromanos , Gorilla gorilla/fisiología , Receptores de Serotonina/efectos de los fármacos , Serotoninérgicos , Animales , Unión Competitiva/efectos de los fármacos , Clonación Molecular , Cobayas , Técnicas In Vitro , Ligandos , Mutación , Receptor de Serotonina 5-HT1D , Receptores de Serotonina/genética , Especificidad de la Especie
5.
J Gen Virol ; 79 ( Pt 5): 1299-307, 1998 May.
Artículo en Inglés | MEDLINE | ID: mdl-9603346

RESUMEN

The RNA genome of grapevine leafroll-associated closterovirus-3 (GLRaV-3) was cloned as a cDNA generated from GLRaV-3-specific dsRNA, and a partial genome sequence of 13154 nucleotides (nt) including the 3' terminus was determined. The sequenced portion contained 13 open reading frames (ORFs) potentially encoding, in the 5'-3' direction, proteins of > 77 kDa (ORF1a; helicase, HEL), 61 kDa (ORF1b; RNA-dependent RNA polymerase, RdRp), 6 kDa (ORF2), 5 kDa (ORF3, small transmembrane protein), 59 kDa (ORF4; heat shock protein 70, HSP70), 55 kDa (ORF5), 35 kDa (ORF6; coat protein, CP), 53 kDa (ORF7; diverged coat protein, CPd), 21 kDa (ORF8), 20 kDa (ORF9), 20 kDa (ORF10), 4 kDa (ORF11), 7 kDa (ORF12), and an untranslated region of 277 nt. ORF1b is probably expressed via a +1 ribosomal frameshift mechanism, most similar to that of lettuce infectious yellows virus (LIYV). Phylogenetic analysis using various gene sequences (HEL, RdRp, HSP70 and CP) clearly demonstrated that GLRaV-3, a mealybug-transmissible closterovirus, is positioned independently from aphid-transmissible monopartite closteroviruses (beet yellows, citrus tristeza and beet yellows stunt) and whitefly-transmissible bipartite closterovirus (lettuce infectious yellows, LIYV). However, another alleged mealybug-transmissible closterovirus, little cherry virus, was shown to be more closely related to the whitefly-transmissible LIYV than to GLRaV-3.


Asunto(s)
Closterovirus/genética , Genoma Viral , ARN Viral , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Viral , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Aminoácido
6.
Gene ; 211(1): 133-40, 1998 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-9573348

RESUMEN

The Cdc7 protein kinase of Saccharomyces cerevisiae is a critical regulator of several aspects of DNA metabolism and cell cycle progression. We describe the isolation of a human gene encoding a Cdc7 homolog. The Cdc7Hs protein sequence is 27% identical to that of the yeast protein, includes features unique to yeast Cdc7, and contains all conserved catalytic residues of protein kinases. The human sequence also shows significant similarity to the cyclin-dependent kinases, in accordance with evidence that yeast Cdc7 is related to the cdks. CDC7Hs is expressed in many normal tissues, but overexpressed in certain tumor types and all transformed cell lines examined. In some of the tumors tested, CDC7Hs expression correlates with expression of a proliferation marker, the histone H3 gene. In other cases, no such correlation was observed. This suggests that CDC7Hs expression may be associated hyperproliferation in some tumors and neoplastic transformation in others.


Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular/química , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Quinasas/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Biomarcadores , División Celular/genética , Clonación Molecular , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Histonas/genética , Humanos , Proteínas Serina-Treonina Quinasas/química , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Células Tumorales Cultivadas
7.
Neurosci Lett ; 235(3): 117-20, 1997 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-9406883

RESUMEN

The 5-HT1D receptor is a potential target of anti-migraine drugs, and here its genes were cloned from chimpanzee, gorilla and rhesus monkey, via polymerase chain reactions with their genomic DNAs and the primers designed from the 5' and 3' untranslated regions of the human receptor. Direct sequencing of the polymerase chain reaction (PCR) products revealed high degrees of identity between their deduced amino acid sequences (the chimpanzee, gorilla and rhesus monkey) and that of human, differing by two, four and 11 residues, respectively. The binding properties of the receptors, as expressed in human embryonic kidney 293 cells, were compared to those obtained with the human and guinea pig receptors, the latter differing by 33 residues from the human receptor. Standard serotonergic ligands including several indoles, ergots and methiothepin bound all the cloned primate and guinea pig receptors with comparable, low nanomolar affinities, leading to high correlation coefficients among their Ki values. R(+)-8-Hydroxydipropylaminotetralin, on the other hand, bound the human receptor with the affinity higher than those for the primates and guinea pig receptors. This indicates that certain chemical templates may differentiate the molecular divergences among the 5-HT1D receptors of various animal species, and the use of the non-human primates will be beneficial for pharmacological characterizations, more relevant to the human receptor, of future novel ligands for the 5-HT1D receptor, which are potential anti-migraine drugs.


Asunto(s)
Cobayas/metabolismo , Primates/metabolismo , Receptores de Serotonina/metabolismo , Animales , Línea Celular , Clonación Molecular , Gorilla gorilla , Humanos , Ligandos , Macaca mulatta , Pan troglodytes , Especificidad de la Especie
8.
Blood ; 89(9): 3457-69, 1997 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-9129054

RESUMEN

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).


Asunto(s)
Huella de ADN , Proteínas de Unión al ADN/metabolismo , Globinas/genética , Filogenia , Animales , Secuencia de Bases , Sitios de Unión , Evolución Biológica , Secuencia Conservada , Embrión de Mamíferos , Feto , Galago , Cabras , Humanos , Ratones , Sondas de Oligonucleótidos , Conejos , Proteínas Represoras/metabolismo , Mapeo Restrictivo , Tiempo
9.
J Biol Chem ; 272(22): 14062-6, 1997 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-9162029

RESUMEN

One basis for the evolution of organisms is the acquisition of new temporal and spatial domains of gene expression. Such novel expression domains could be generated either by cis sequence changes that alter the complement of trans-acting regulators binding to control elements or by changes in the expression patterns of one or more of the regulatory (trans) factors themselves. The gamma globin gene is a prime example of a gene that has undergone a distinct change in temporal expression at a defined time in evolution. Approximately 35-55 million years ago, the previously embryonic gamma gene acquired a fetal expression pattern. This change occurred in a simian primate ancestor after the separation of simian and prosimian primates but before the further separation of the major simian lineages; thus, the (prosimian) galago gamma gene retains the ancestral embryonic expression pattern, whereas the (simian) human gamma gene is fetal. This analysis of galago and human gamma genes in transgenic mice demonstrates that cis changes in sequences within a 4.0-kilobase region surrounding the gamma gene were responsible for the evolution of a novel fetal expression pattern in the gamma globin genes of simian primates.


Asunto(s)
Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Animales , Femenino , Galago , Humanos , Ratones , Ratones Transgénicos , Embarazo
10.
Gene ; 188(1): 9-16, 1997 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-9099852

RESUMEN

Detailed analyses of transcripts encoding various isoforms of the human potassium (K+, inward rectifying) channel ROM-K (also referred to as K(ir)1.1) revealed the existence of at least five distinct transcripts [Shuck et al., J. Biol. Chem. 269 (1994) 24261-24270]. These five hROM-K transcripts appear to be the result of alternative splicing of five exons. The nucleotide sequence of the genomic DNA including and spanning these exons (the KCNJ1 locus) was obtained directly from lambda and P1 clones (a total of 40 kb). The organization of the hKCNJ1 gene was determined by combining this sequence information with data obtained from primer extension and RT-PCR experiments. It appears that the hKCNJ1 gene utilizes multiple promoters, with promoter-like elements found 5' of exons 1, 4, or 5. The promoter 5' of exon 5 was unexpected; thus, it appears that the hKCNJ1 gene is capable of producing six distinct hROM-K transcripts via the use of three promoters and alternative splicing of five exons. Comparisons of the rat and human ROM-K cDNA sequences find human homologs (orthologs) for two of the three distinct rROM-K transcripts. A search of the complete human KCNJ1 sequence with the exon sequence that defines the other rROM-K transcript located a region of shared nucleotides, a putative sixth exon, in the hKCNJ1 gene. This finding suggests that the rKCNJ1 gene may contain an exon that is no longer or infrequently used in transcripts derived from the hKCNJ1 gene.


Asunto(s)
Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN , Cartilla de ADN , Exones , Humanos , Datos de Secuencia Molecular , Poli A , Regiones Promotoras Genéticas , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN
11.
Genomics ; 39(1): 90-4, 1997 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-9027490

RESUMEN

The locus control region (LCR) of mammalian beta-globin genes covers at least 17 kb at the 5' end of the gene cluster and has been implicated in chromatin domain opening, enhancement, and insulation from neighboring sequences. Functional dissection of the LCR has defined the minimal cores for four of the five major DNase hypersensitive sites (HSs) that mark this regulatory region. To examine fully the patterns of conserved sequences in the mammalian homologs to the beta-globin LCR, we determined the complete DNA sequence of the galago beta-globin LCR and completed previously unsequenced regions of the rabbit LCR. Simultaneous alignment of these sequences with the human, goat, and mouse LCRs revealed conserved sequences (phylogenetic footprints) detected using three largely independent methods. The most highly conserved segments are found both within the HS cores and in some but not all regions flanking the cores. These results argue for an extended pattern of well-conserved sequences, many of which lie outside the minimal cores, and we show that a key sequence required for domain opening by the region including HS3 maps about 1 kb 5' to the minimal core. Differential phylogenetic footprints, containing sequences conserved in nonhuman mammals but not in humans, are found primarily around HS3, consistent with some species-specific differences in function that may be important for differences in hemoglobin switching during development.


Asunto(s)
Secuencia Conservada , Globinas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Mapeo Cromosómico , Desoxirribonucleasas/metabolismo , Galago , Humanos , Ratones , Datos de Secuencia Molecular , Conejos , Alineación de Secuencia
12.
J Immunol ; 158(2): 748-55, 1997 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-8992991

RESUMEN

The objective of these studies was to examine the molecular mechanisms involved in transcriptional regulation of the gene for the intracellular structural variant of the IL-1 receptor antagonist (icIL-1Ra) molecule. By reverse transcription-PCR analysis, constitutive expression of endogenous icIL-1Ra mRNA was observed in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937, or in the lymphocyte cell lines Raji and Jurkat. However, icIL-1Ra mRNA expression was observed in response to stimulation with LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. To examine the mechanisms of transcriptional regulation, 4.5 kb of the 5' flanking sequence was isolated from the human icIL-1Ra gene, sequenced, cloned into a luciferase expression vector (pIC4525.Luc), and examined in transfection studies. The pIC4525.Luc construct exhibited a pattern of expression in epithelial and macrophage cell lines similar to that of the endogenous icIL-1Ra gene. To obtain a generalized map of cell type-specific and inducible cis-acting DNA elements, nested 5' deletional mutants of the icIL-1Ra promoter were constructed. Results from transfection studies with these icIL-1Ra promoter/luciferase fusion constructs indicated that constitutive expression in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, -288 to -156, and -156 to -49. In contrast, basal expression of pIC4525.Luc in transfected but unstimulated RAW 264.7 cells was under the control of a weak inhibitory region located between bases -4525 to -1438 and a strong positive element between -156 and -49. LPS induction of icIL-1Ra transcription in RAW 264.7 cells was regulated by strong positively acting DNA regions between bases -1438 to -909 and -156 to -49. In summary, the proximal region of the icIL-1Ra promoter, between bases -156 to -49, contains positive cis-acting elements that are needed for expression in both epithelial and monocyte cell lines. However, our results indicate that the ability of this proximal promoter region to control expression is strongly influenced, both positively and negatively, by other upstream cis-acting elements in a cell type-specific manner.


Asunto(s)
Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/antagonistas & inhibidores , Sialoglicoproteínas/biosíntesis , Secuencia de Aminoácidos , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Células Jurkat , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Sialoglicoproteínas/clasificación , Sialoglicoproteínas/efectos de los fármacos , Sialoglicoproteínas/genética , Células Tumorales Cultivadas
13.
J Biol Chem ; 272(1): 586-93, 1997 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-8995301

RESUMEN

The DNA sequence encoding the rat brain inward rectifier-10 K+ channel was amplified from rat brain RNA using reverse transcription-polymerase chain reaction and used to clone the human homolog. Low stringency screening of a human kidney cDNA library and subsequent DNA sequence analysis identified two related K+ inward rectifier cDNAs, referred to as Kir1.2 and Kir1.3, which were derived from transcription of distinct human genes. Kir1.2 represents the human homolog of the rat BIRK-10 sequence, whereas Kir1.3 was unique compared with all available sequence data bases. The genes that encode Kir1.2 and Kir1.3 were mapped to human chromosomes 1 and 21, respectively. Both genes showed tissue-specific expression when analyzed by Northern blots. Kir1.2 was only detected in brain >> kidney and was detected at high levels in all brain regions examined. Kir1.3 was most readily detected in kidney and was also expressed in pancreas > lung. Comparative analysis of the predicted amino acid sequences for Kir1.2 and Kir1.3 revealed they were 62% identical. The most remarkable difference between the two polypeptides is that the Walker Type A consensus binding motif present in both Kir1.1 and Kir1.2 was not conserved in the Kir1.3 sequence. Expression of the Kir1.2 polypeptide in Xenopus oocytes resulted in the synthesis of a K+-selective channel that exhibited an inwardly rectifying current-voltage relationship and was inhibited by external Ba2+ and Cs+. Kir1.2 current amplitude was reduced by >85% when the pH was decreased from pH 7.4 to 5.9 using the membrane-permeant buffer acetate but was relatively unaffected when pH was similarly lowered using membrane-impermeant biphthalate. The inhibition by intracellular protons was voltage-independent with an IC50 of pH 6.2 and a Hill coefficient of 1.9, suggesting the cooperative binding of 2 protons to the intracellular face of the channel. In contrast, Kir1.3 expression in Xenopus oocytes was not detectable despite the fact that the cRNA efficiently directed the synthesis of a polypeptide of the expected Mr in an in vitro translation system. Co-expression of Kir1.3 with either Kir1.1 or Kir1.2 reduced currents resulting from expression of these inward-rectifier subunits alone, consistent with a dominant negative influence on Kir1.1 and Kir1.2 expression.


Asunto(s)
Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Secuencia de Aminoácidos , Animales , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 21 , Clonación Molecular , Conductividad Eléctrica , Humanos , Concentración de Iones de Hidrógeno , Riñón , Glicoproteínas de Membrana/química , Potenciales de la Membrana , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Distribución Tisular
14.
Arch Virol ; 142(6): 1101-16, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9229001

RESUMEN

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.


Asunto(s)
Cápside/genética , Closterovirus/genética , Agrobacterium tumefaciens , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario , ADN Viral , Frutas/virología , Biblioteca de Genes , Ingeniería Genética , Vectores Genéticos , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Plantas Tóxicas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Nicotiana , Transformación Genética
15.
Gene ; 205(1-2): 47-57, 1997 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-9461379

RESUMEN

Phylogenetic reconstructions by parsimony were carried out on an enlarged body of primate gamma1 and gamma2-globin sequences. The results confirm that gamma1 and gamma2 arose from a tandem duplication in an ancient simian lineage ancestral to both platyrrhines (New World monkeys) and catarrhines (Old World monkeys and hominoids). Gene conversions between the two gamma homologs were frequent over the gamma gene proper but less frequent over the 5' flanking and very infrequent over the 3' flanking regions. The ancient platyrrhine conversion in the most distal 5' flanking region had the polarity of gamma2-->gamma1. Recent platyrrhine conversions between 5' regulatory sequences were very infrequent, in striking contrast to catarrhines which have large, uninterrupted stretches of converted 5' regulatory sequences. Comparisons of reconstructed ancestral primate and simian gamma promoter sequences revealed an accumulation of 21 nucleotide substitutions concentrated in or near cis-elements that may have mediated the change from embryonic to fetal gamma expression. Almost all 21 substitutions were retained in the lineages leading to functional gamma genes of extant catarrhines (both gamma1 and gamma2) and platyrrhines (most often gamma2). Fewer of these simian specific substitutions were retained in the platyrrhine gamma1 genes and new mutations occurred more often in the platyrrhine gamma1 than gamma2 promoters.


Asunto(s)
Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Animales , Secuencia de Bases , ADN , Feto , Conversión Génica , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Primates , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico
16.
Gene ; 205(1-2): 73-94, 1997 Dec 31.
Artículo en Inglés | MEDLINE | ID: mdl-9461381

RESUMEN

Locus control regions (LCRs) are cis-acting DNA segments needed for activation of an entire locus or gene cluster. They are operationally defined as DNA sequences needed to achieve a high level of gene expression regardless of the position of integration in transgenic mice or stably transfected cells. This review brings together the large amount of DNA sequence data from the beta-globin LCR with the vast amount of functional data obtained through the use of biochemical, cellular and transgenic experimental systems. Alignment of orthologous LCR sequences from five mammalian species locates numerous conserved regions, including previously identified cis-acting elements within the cores of nuclease hypersensitive sites (HSs) as well as conserved regions located between the HS cores. The distribution of these conserved sequences, combined with the effects of LCR fragments utilized in expression studies, shows that important sites are more widely distributed in the LCR than previously anticipated, especially in and around HS2 and HS3. We propose that the HS cores plus HS flanking DNAs comprise a 'unit' to which proteins bind and form an optimally functional structure. Multiple HS units (at least three: HS2, HS3 and HS4 cores plus flanking DNAs) together establish a chromatin structure that allows the proper developmental regulation of genes within the cluster.


Asunto(s)
Globinas/genética , Región de Control de Posición/genética , Familia de Multigenes , Filogenia , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad de la Especie
17.
Hum Mol Genet ; 5(8): 1149-53, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8842734

RESUMEN

Mutations in the dystrophin gene are responsible for Duchenne and Becker muscular dystrophy (DMD/BMD). Studies of dystrophin expression and function have benefited from use of the mdx mouse, an animal model for DMD/BMD. Here we characterized mutations in three additional strains of mdx mice, the mdx2cv, mdx4cv and mdx5cv alleles. The mutation in the mdx2cv mouse was found to be a single base change in the splice acceptor sequence of dystrophin intron 42. This mutation leads to a complex pattern of aberrant splicing that generates multiple transcripts, none of which preserve the normal open reading frame. In the mdx5cv allele, the dystrophin mRNA contains a 53 bp deletion of sequences from exon 10. Analysis of the genomic DNA uncovered a single A to T transversion in exon 10. Although this base change does not alter the encoded amino acid, a new splice donor was created (GTGAG) that generates a frameshifting deletion in the processed mRNA. In the mdx4cv allele, direct sequencing revealed a C to T transition in exon 53, creating an ochre codon (CAA to TAA). The differential location of these mutations relative to the seven known dystrophin promoters results in a series of mdx mouse mutants that differ in their repertoire of isoform expression, such that these mice should be useful for studies of dystrophin expression and function. The mdx4cv and mdx5cv strains may be of additional use in gene transfer studies due to their low frequency of mutation reversion.


Asunto(s)
Distrofina/genética , Ratones Endogámicos mdx/genética , Distrofia Muscular Animal/genética , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Mutación del Sistema de Lectura , Expresión Génica , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Empalme del ARN/genética
18.
Proc Natl Acad Sci U S A ; 93(13): 6510-5, 1996 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-8692846

RESUMEN

Nucleotide sequences were determined for the gamma1- and gamma2-globin loci from representatives of the seven anciently separated clades in the three extant platyrrhine families (Atelidae, Pitheciidae, and Cebidae). These sequences revealed an evolutionary trend in New World monkeys either to inactivate the gamma1 gene or to fuse it with the gamma2 gene, i.e. to have only one functional fetally expressed gamma gene. This trend is clearly evident in six of the seven clades: (i) it occurred in atelids by deletion of most of the gamma1 gene in the basal ancestor of this clade; (ii-iv) in pitheciid titi, saki, and cebid capuchin monkeys by potentially debilitating nucleotide substitutions in the proximal CCAAT box of the gamma1 promoters and (v and vi) in cebid owl and squirrel monkeys by crossovers that fused 5' sequence from gamma1 with 3' sequence from gamma2. In the five clades with gamma1 and gamma2 loci separated by intergenic sequences (the fifth clade being the cebid marmosets), the gamma2 genes retained an unaltered proximal CCAAT motif and their gamma2 promoters accumulated fewer nucleotide substitutions than did the gamma1 promoters. Thus, phylogenetic considerations indicate that the stem platyrrhines, ancestral to all New World monkeys, had gamma2 as the primary fetally expressed gamma gene. A further inference is that when the earlier stem anthropoid gamma gene duplicated, gamma2 (at its greater downstream distance from epsilon) could evade embryonic activation by the locus control region but could be fetally activated once released by regulatory mutations from fetal repressors.


Asunto(s)
Evolución Biológica , Cebidae/genética , Globinas/genética , Animales , Secuencia de Bases , Clonación Molecular , Intercambio Genético , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Recombinación Genética , Homología de Secuencia de Ácido Nucleico
19.
Glycobiology ; 6(2): 231-41, 1996 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8727794

RESUMEN

We have previously characterized a cDNA that encodes a full length human UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase (GalNAc-transferase) (J.A. Meurer et al., J. Biochem., 118, 568-574, 1995). The present report describes the characterization of the corresponding human GalNAc-transferase gene and a related pseudogene. Two human genomic libraries, lambda and P1, were screened with probes derived from the human GalNAc-transferase nucleotide sequence, resulting in the isolation of four genomic clones. Southern blotting, PCR analysis, and sequencing revealed that three clones, lambda.HG-5, P1.GALN-A, and P1.GALN-B, contained overlapping genomic sequences that encompass over 55 kilobase pairs (kb) of genomic DNA and comprise a portion of the human GalNAc-transferase 5'-and 3'-untranslated regions and the entire coding region. The human GalNAc-transferase gene structure consists of at least 11 exons ranging in size from 99 to > 620 nucleotides which are separated by 10 introns ranging in size from 0.7 to approximately 12.5 kb. The fourth genomic clone, P1-GALN-psi, contained a approximately 2.4 kb sequence region which shares an overall 78.6% nucleotide identity with coding region exons 1 and 3 through 11 of the human GalNAc-transferase gene. However, a lack of intron sequences, as well as the presence of multiple nucleotide mutations, insertions, and deletions that disrupt the potential GalNAc-transferase reading frame, suggest that P1.GALN-psi contains a processed pseudogene. Screening of a human/rodent somatic cell hybrid panel with a P1.GALN-psi probe localized the GalNAc-transferase pseudogene to chromosome 3. Hence, the human genome contains at least two related GalNAc-transferase genes that are located on separate chromosomes.


Asunto(s)
N-Acetilgalactosaminiltransferasas/genética , Seudogenes , Secuencia de Bases , Southern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 3 , Clonación Molecular , ADN Complementario , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Polipéptido N-Acetilgalactosaminiltransferasa
20.
Mol Phylogenet Evol ; 5(1): 18-32, 1996 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8673285

RESUMEN

We describe three strategies for the identification of specific cis and trans factors that regulate globin gene expression, all three of which are based on the evolution of the globin genes and their expression patterns. The first approach, phylogenetic footprinting, relies on a search for sequence similarities and is designed to elucidate the factors that control those expression patterns which are shared by orthologous globin genes of all eutherian mammals (e.g., the expression of the epsilon globin genes in the embryonic yolk sac and its repression in fetal and adult hematopoietic tissues). The second approach, differential phylogenetic footprinting, relies on a search for sequence differences. This approach may be of value in identifying the mechanisms underlying the generation of novel expression patterns in specific lineages (e.g., the expression of gamma as a fetal gene in the simian primates in contrast with the embryonic expression of gamma in all other mammals). Finally, motif-based phylogenetic analysis takes into consideration the fact that many transcription factors are quite flexible in the recognition of their cognate sites. The approach allows the detection of functionally conserved binding sites despite their sequence variation.


Asunto(s)
Evolución Molecular , Genes de Cambio , Globinas/genética , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , ADN/genética , Huella de ADN , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Mamíferos , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Factores de Transcripción/genética
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